Toxigenic Vibrio cholerae belonging to the O1 and O139 serogroups cause cholera, an acute, waterborne diarrheal disease that causes seasonal epidemics in many developing countries such as Bangladesh. Although transmission of cholera typically occurs through water contaminated with the pathogenic organisms, the natural mechanisms that facilitate the epidemic spread of cholera and support the survival of the pathogen in water between seasonal epidemics in Bangladesh are not clear. We will apply a new method we developed called AST to monitor and study the nature of "conditionally viable environmental cells (CVEC)" of pathogenic V. cholerae that exist in surface waters of Bangladesh. Because CVEC are likely derived from biofilm-like aggregates of V. cholerae shed in human cholera stools, we propose a broad program to understand the infectious form of V. cholerae in cholera stools, and their survival in the aquatic environment. We will isolate CVEC from environmental waters and characterize them metabolically, morphologically, immunochemically, and for their innate infectivity in animal models. Because we have shown phage predation apparently ends cholera epidemics, we will determine whether CVEC of V. cholerae are protected from phage predation due to their possible low metabolic activity or physical properties. Our proposed analysis will include biochemical and genetic studies designed to understand enhanced transmissibility of stool-derived vibrios, their environmental survival as CVEC and resuscitation of CVEC to pathogenic viable organisms. We propose to produce CVEC-like V. cholerae cells from cholera stools (CVEC-H), or from cells shed by experimentally infected rabbits (CVEC-R) or under fully laboratory conditions (CVEC-L). To understand the genetic basis for the transition between various cell types, we will conduct microarray-based, genomic transcriptional analysis. Standard genetic methods will be utilized to identify genes involved in formation of CVEC-R and CVEC-L. A library of 2623 defined transposon insertion mutants of V. cholerae will aid in the systematic identification of genes involved in low sodium osmo-stress. Together with data from environmental monitoring, these studies should provide a better understanding of the infectious cycle of cholera and thus inspire new effective interventions for interrupting the natural history of this disease in developing countries. [unreadable] [unreadable] [unreadable]